Regulation of gene function occurs by several mechanisms in eukaryotic cells. Amongst these mechanisms are gene transcription regulation, mRNA translation regulation, and post-translation modification of proteins. Post-translation modification of proteins includes several processes whereby proteins are covalently altered to affect cellular, sub-cellular localization, stability, transport, interaction specificity, enzymatic activity, and numerous other characteristics.
Common and extensively studied covalent modification processes include acetylation, glycosylation, and phosphorylation. Less well characterized is a process that involves the covalent addition of polymers of ADP-ribose to protein targets. The polymer is termed “poly(ADP-ribose),” and the enzyme(s) responsible for this activity have been variously called poly(ADP-ribose) polymerase (PARP), poly(ADP-ribose) synthetase (PARS), or ADP-ribosyl transferase (ADPRT) [Althaus and Richter, ADP-Ribosylation of Proteins: Enzymology and Biochemical Significance, Molecular Biochemistry and Biophysics, Springer-Verlag (1987)]. A previously identified PARP gene product (hereinafter “PARP1”) is expressed at high levels in the nuclei of cells and is dependent upon DNA damage for activation [Szabó and Dawson, Trends Pharmacol Sci 19(7):287–98 (1998)]. Current models hypothesize that PARP1 binds to DNA single or double stranded breaks through an amino terminal DNA binding domain. The binding activates the carboxy terminal catalytic domain and results in the formation of polymers of ADP-ribose on target molecules. PARP1 is itself a target of polyADP-ribosylation by virtue of a centrally located automodification domain. The ribosylation of PARP1 causes dissociation of the PARP1 molecules from the DNA. The entire process of binding, ribosylation, and dissociation occurs very rapidly. It has been suggested that this transient binding of PARP1 to sites of DNA damage may result in the recruitment of DNA repair machinery or may act to suppress recombination long enough for the recruitment of repair machinery [Satoh and Lindahl, Nature 356(6367):356–8 (1992)].
The source of ADP-ribose for the PARP reaction is nicotinamide adenine dinucleotide (NAD). NAD is synthesized in cells from cellular ATP stores and thus high levels of activation of PARP activity can rapidly lead to depletion of cellular energy stores. It has been demonstrated that induction of PARP activity can lead to cell death that is correlated with depletion of cellular NAD and ATP pools [Yamamoto et al., Nature 294(5838):284–6 (1981); Sims et al., Biochemistry 22(22):5188–94 (1983)]. PARP activity is induced in many instances of oxidative stress or during inflammation. For example, during reperfusion of ischemic tissues reactive nitric oxide is generated and nitric oxide results in the generation of additional reactive oxygen species including hydrogen peroxide, peroxynitrate and hydroxyl radical [Szabó, Eur J Pharmacol 350(1):1–19 (1998)]. These latter species can directly damage DNA and the resulting damage induces activation of PARP activity. Frequently, it appears that sufficient activation of PARP activity occurs so that the cell energy stores are depleted and the cell dies.
A similar mechanism is believed to operate during inflammation when endothelial cells and pro-inflammatory cells synthesize nitric oxide which results in oxidative DNA damage in surrounding cells and the subsequent activation of PARP activity [Szabó 1998, supra]. Such a mechanism is also believed to play a role in tissue damage associated with transient cerebral ischemia, in which excessive NMDA receptor activation mediates DNA damage, which induces excessive PARP activation [Lo et al., Stroke 29:830–6 (1998)]. The cell death that results from PARP activation is believed to be a major contributing factor in the extent of tissue damage that results from ischemia/reperfusion injury or from inflammation.
Two lines of evidence suggest that PARP activity is a critical element in those processes. First, chemical inhibitors of PARP activity have been successfully used to reduce tissue damage resulting in animal models of ischemia/reperfusion injury or inflammation. Second, mice in which both alleles of PARP1 have been disabled (PARP1 knockout mice) are resistant to numerous forms of ischemia/reperfusion injury and detrimental effects of inflammation. Because of those observations, potent small molecule inhibitors of PARP activity have great potential as clinical drug candidates in several indications.
The experimental data derived from the PARP1 knockout mice suggest that inhibitors of PARP1 function may be clinically beneficial. However, a recent report demonstrates that PARP1 knockout mice are not devoid of DNA damage-inducible PARP activity [Shieh et al., J Biol Chem 273(46):30069–72 (1998)]. A recently identified gene product, tankyrase, has been shown to have poly(ADP) ribosylation activity [Smith et al., Science 282(5393):1484–7 (1998)]. Tankyrase activity however, does not appear to be inducible by DNA damage and thus is unlikely to account for the activity observed in PARP1 knockout mice. It has been suggested that the residual DNA damage induced PARP activity in PARP1 knockout mice may be due to the activity of a second PARP gene, which has been identified in the mouse and named murine PARP2 [Shieh et al. (1998), supra]. The existence of multiple PARP genes in mammals suggests that appropriate drug design for human therapeutics requires the identification of additional human gene products with PARP activity. A gene comparable to mouse PARP2 has not previously been identified in humans.
In view of the above considerations, it is clear that existing knowledge is lacking with respect to cellular DNA repair mechanisms, signaling and induction of cell death in response to DNA damage, mechanisms of inflammation, and treatment of inflammation-mediated disease states. Thus, there exists a need in the art for the identification of additional human PARP-like molecules for use in determining the selectivity of therapeutics designed to inhibit PARP function and as targets in their own right for therapeutic intervention in human diseases. The profiling of PARP inhibitors on additional PARP gene products may allow for the PARP-selective drugs, which could be beneficial for particular indications, the reduction of undesirable side effects, or the targeting of therapeutics to selected tissues. Likewise, the identification of hPARP2 will allow for the development of hPARP2 specific therapeutics, which may also have benefits in terms of particular disease indications, the reduction of undesirable side effects, and the targeting of therapeutics to particular tissues. The identification of human hPARP2 would also allow for the development of drugs with the ability to inhibit both PARP activities that may also have therapeutic benefit. Other purposes and advantages of the invention will be readily apparent to the artisan having ordinary skill in the art.